SummaryProteinase inhibitor II proteins (PIN2) are serine proteinase inhibitors found in the Solanaceae. Here, we assign functions in seed development to two Solanum americanum genes, SaPIN2a and SaPIN2b, encoding proteinase inhibitor II. Their mRNAs and proteins have been previously localized to the reproductive tissues, including the inner cell layers of ovules in senescent flowers at the beginning of fruit development, suggestive of their endogenous roles in reproductive development. We have employed RNA interference (RNAi)-induced post-transcriptional gene silencing (PTGS) to further investigate the role of SaPIN2a and SaPIN2b during seed development. A SaPIN2a-derived construct that shared 83% nucleotide homology to SaPIN2b was used in PTGS to silence both genes. Northern blot analyses confirmed that the PIN2-RNAi transgenic plants contain small interfering RNAs (siRNAs) and exhibit reduced levels of SaPIN2a and SaPIN2b mRNAs at various stages of floral development. A reduction in seed set due to seed abortion was observed in PIN2-RNAi transgenic lines. Cytological and molecular analyses of these lines showed the lack of SaPIN2a and SaPIN2b mRNAs and proteins at the inner cell layers of the ovules in senescent flowers. Aborted seeds in transgenic fruits had an abnormal endothelium. The anomalous expansion of the endothelium prevented proper development of the endosperm and embryo, leading to seed abortion. Our observations indicate that SaPIN2a and SaPIN2b are essential for seed development and suggest that the endothelium may protect the embryo sac, allowing proper formation of the endosperm and embryo, as a result of its ability to produce proteinase inhibitors.

SummaryProteinase inhibitor II (PIN2) proteins from the Solanaceae family have been previously used in plant transformation to acquire protection against caterpillars. Some of these PIN2 proteins have been shown to exhibit exogenous activities against trypsin and/or chymotrypsin in vitro. Despite their application in conferring insect resistance in transgenic plants, the endogenous roles of this family of proteins in various plant species have not been well defined. To investigate the exogenous and endogenous functions of PIN2 proteins, cDNAs encoding PIN2 proteins from the weed Solanum americanum (American black nightshade), designated SaPIN2a and SaPIN2b, were cloned and characterized. The localization of S. americanum SaPIN2a and SaPIN2b mRNAs and proteins in the reproductive tissues destined to undergo developmental programmed cell death subsequently led to investigations into their function during seed development. Using plant transformation of lettuce and S. americanum, it was evident that: (1) the expression of SaPIN2a in transgenic lettuce conferred resistance to cabbage looper (Trichoplusia ni) caterpillars; and (2) the expression of siRNAs from a PIN2-RNAi construct resulted in transgenic S. americanum that were impaired in seed development. These results suggest that S. americanum PIN2 proteins not only enhance resistance to caterpillars (when expressed exogenously) but they function in inhibiting endogenous proteases that are expressed during seed development. Specifically, the aborted seeds of PIN2-RNAi lines showed abnormal endothelium that subsequently affected endosperm and embryo development.

The heterologous expression of serine proteinase inhibitor II (PIN2) proteins confers insect resistance in transgenic plants, but little is known of their endogenous roles. We have cloned two cDNAs encoding Solanum americanum PIN2 proteins, SaPIN2a and SaPIN2b. SaPIN2a is highly expressed in stem, particularly in the phloem, suggesting it could possibly regulate proteolysis in the sieve elements. When SaPIN2a was expressed in transgenic lettuce, we observed an inhibition of endogenous trypsin- and chymotrypsin-like activities. Here, we demonstrate that both SaPIN2a and SaPIN2b are expressed in floral tissues that are destined to undergo developmental programmed cell death (PCD), suggesting possible endogenous roles in inhibiting trypsin- and chymotrypsin-like activities during flower development. Northern and western blot analyses revealed that SaPIN2a and SaPIN2b mRNAs and proteins show highest expression early in floral development. In situ hybridization analysis and immunolocalization on floral sections, localized SaPIN2a and SaPIN2b mRNAs and their proteins to tissues that would apparently undergo PCD: the ovules, the stylar transmitting tissue, the stigma and the vascular bundles. Detection of PCD in floral sections was achieved using terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) analysis. Examination of the mid-style before, and 1 day after, pollination revealed that high expression of SaPIN2a and SaPIN2b in the style was inversely correlated with PCD.